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1.
Nature ; 574(7779): 571-574, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645724

RESUMO

To safeguard genome integrity in response to DNA double-strand breaks (DSBs), mammalian cells mobilize the neighbouring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes1. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin-CST-POLα complex2. How this pathway reflects and influences the three-dimensional nuclear architecture is not known. Here we use super-resolution microscopy to show that 53BP1 and RIF1 form an autonomous functional module that stabilizes three-dimensional chromatin topology at sites of DNA breakage. This process is initiated by accumulation of 53BP1 at regions of compact chromatin that colocalize with topologically associating domain (TAD) sequences, followed by recruitment of RIF1 to the boundaries between such domains. The alternating distribution of 53BP1 and RIF1 stabilizes several neighbouring TAD-sized structures at a single DBS site into an ordered, circular arrangement. Depletion of 53BP1 or RIF1 (but not shieldin) disrupts this arrangement and leads to decompaction of DSB-flanking chromatin, reduction in interchromatin space, aberrant spreading of DNA repair proteins, and hyper-resection of DNA ends. Similar topological distortions are triggered by depletion of cohesin, which suggests that the maintenance of chromatin structure after DNA breakage involves basic mechanisms that shape three-dimensional nuclear organization. As topological stabilization of DSB-flanking chromatin is independent of DNA repair, we propose that, besides providing a structural scaffold to protect DNA ends against aberrant processing, 53BP1 and RIF1 safeguard epigenetic integrity at loci that are disrupted by DNA breakage.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Instabilidade Genômica , Conformação de Ácido Nucleico , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
2.
Cancer Sci ; 110(11): 3543-3552, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31541611

RESUMO

Poly ADP-ribose polymerase inhibitors (PARPi) have shown promising therapeutic efficacy in triple-negative breast cancer (TNBC) patients. However, resistance ultimately develops, preventing a curative effect from being attained. Extensive investigations have indicated the diversity in the mechanisms underlying the PARPi sensitivity of breast cancer. In this study, we found that DNA damage binding protein 2 (DDB2), a DNA damage-recognition factor, could protect TNBC cells from PARPi by regulating DNA double-strand break repair through the homologous recombination pathway, whereas the depletion of DDB2 sensitizes TNBC cells to PARPi. Furthermore, we found that DDB2 was able to stabilize Rad51 by physical association and disrupting its ubiquitination pathway-induced proteasomal degradation. These findings highlight an essential role of DDB2 in modulating homologous recombination pathway activity and suggest a promising therapeutic target for TNBC.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Linhagem Celular Tumoral , Reparo do DNA , Proteínas de Ligação a DNA/deficiência , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Proteínas de Neoplasias/metabolismo , Ubiquitinação
3.
Nucleic Acids Res ; 47(16): 8537-8547, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31226203

RESUMO

Apurinic/apyrimidinic (AP) sites are a class of highly mutagenic and toxic DNA lesions arising in the genome from a number of exogenous and endogenous sources. Repair of AP lesions takes place predominantly by the base excision pathway (BER). However, among chemically heterogeneous AP lesions formed in DNA, some are resistant to the endonuclease APE1 and thus refractory to BER. Here, we employed two types of reporter constructs accommodating synthetic APE1-resistant AP lesions to investigate the auxiliary repair mechanisms in human cells. By combined analyses of recovery of the transcription rate and suppression of transcriptional mutagenesis at specifically positioned AP lesions, we demonstrate that nucleotide excision repair pathway (NER) efficiently removes BER-resistant AP lesions and significantly enhances the repair of APE1-sensitive ones. Our results further indicate that core NER components XPA and XPF are equally required and that both global genome (GG-NER) and transcription coupled (TC-NER) subpathways contribute to the repair.


Assuntos
Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Proteína de Xeroderma Pigmentoso Grupo A/genética , Sequência de Bases , Proteína 9 Associada à CRISPR/genética , Proteína 9 Associada à CRISPR/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular Transformada , DNA/química , DNA/metabolismo , Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Proteínas de Ligação a DNA/deficiência , Fibroblastos/citologia , Fibroblastos/metabolismo , Edição de Genes/métodos , Técnicas de Inativação de Genes , Genoma Humano , Humanos , Mutação , Ligação Proteica , Pele/citologia , Pele/metabolismo , Transcrição Genética , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo
4.
Nat Commun ; 10(1): 2771, 2019 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-31235807

RESUMO

Diverse antibody repertoires are generated through remote genomic interactions involving immunoglobulin variable (VH), diversity (DH) and joining (JH) gene segments. How such interactions are orchestrated remains unknown. Here we develop a strategy to track VH-DHJH motion in B-lymphocytes. We find that VH and DHJH segments are trapped in configurations that allow only local motion, such that spatially proximal segments remain in proximity, while spatially remote segments remain remote. Within a subset of cells, however, abrupt changes in VH-DHJH motion are observed, plausibly caused by temporal alterations in chromatin configurations. Comparison of experimental and simulated data suggests that constrained motion is imposed by a network of cross-linked chromatin chains characteristic of a gel phase, yet poised near the sol phase, a solution of independent chromatin chains. These results suggest that chromosome organization near the sol-gel phase transition dictates the timing of genomic interactions to orchestrate gene expression and somatic recombination.


Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Regulação da Expressão Gênica/fisiologia , Genes de Imunoglobulinas/genética , Recombinação V(D)J/fisiologia , Animais , Linfócitos B/metabolismo , Linhagem Celular , Cromossomos/genética , Proteínas de Ligação a DNA/deficiência , Genômica , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Camundongos , Transição de Fase
5.
Mol Biol (Mosk) ; 53(2): 274-281, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31099777

RESUMO

Expression of Saccharomyces cerevisiae proteasomal genes is regulated in a coordinated manner by a system that includes the ScRpn4 transcription factor and its binding site known as PACE. Earlier we showed that, Rpn4-like proteins from the biotechnologically important yeast species Komagataella pfaffii (Pichia pastoris), Yarrowia lipolytica, and Debaryomyces hansenii are capable of complementing the RPN4 deletion in S. cerevisiae in spite of their low structural similarity to ScRpn4. The opportunistic yeast pathogen Candida glabrata has a gene coding for a Rpn4-like protein, which has not been characterized experimentally yet. The С. glabrata ortholog ScRpn4 was expressed heterologously and found to restore the stress resistance and expression of proteasomal genes in a mutant S. cerevisiae strain with a RPN4 deletion. This complementation required the unique N-terminal region of CgRpn4. The results indicate that CgRpn4 acts as a transcriptional activator of proteasomal genes. The S. cerevisiae model can be used for further structural and functional analyses of CgRpn4.


Assuntos
Candida glabrata/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Deleção de Genes , Teste de Complementação Genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Regulação Fúngica da Expressão Gênica , Saccharomyces cerevisiae/metabolismo
6.
Nucleic Acids Res ; 47(8): 4086-4110, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30986824

RESUMO

Ataxia with oculomotor apraxia type 1 (AOA1) is an early onset progressive spinocerebellar ataxia caused by mutation in aprataxin (APTX). APTX removes 5'-AMP groups from DNA, a product of abortive ligation during DNA repair and replication. APTX deficiency has been suggested to compromise mitochondrial function; however, a detailed characterization of mitochondrial homeostasis in APTX-deficient cells is not available. Here, we show that cells lacking APTX undergo mitochondrial stress and display significant changes in the expression of the mitochondrial inner membrane fusion protein optic atrophy type 1, and components of the oxidative phosphorylation complexes. At the cellular level, APTX deficiency impairs mitochondrial morphology and network formation, and autophagic removal of damaged mitochondria by mitophagy. Thus, our results show that aberrant mitochondrial function is a key component of AOA1 pathology. This work corroborates the emerging evidence that impaired mitochondrial function is a characteristic of an increasing number of genetically diverse neurodegenerative disorders.


Assuntos
Proteínas de Ligação a DNA/genética , GTP Fosfo-Hidrolases/genética , Mitocôndrias/genética , Degradação Mitocondrial/genética , Proteínas Nucleares/genética , Ataxias Espinocerebelares/congênito , Linhagem Celular Transformada , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/deficiência , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Complexo de Proteínas da Cadeia de Transporte de Elétrons/metabolismo , GTP Fosfo-Hidrolases/deficiência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Linfócitos/metabolismo , Linfócitos/patologia , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Nucleares/deficiência , Análise de Sequência com Séries de Oligonucleotídeos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação Oxidativa , Transdução de Sinais , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/metabolismo , Ataxias Espinocerebelares/patologia
7.
Appl Microbiol Biotechnol ; 103(10): 4103-4112, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30953122

RESUMO

SpoIIID is a small, sequence-specific DNA-binding protein which can direct many genes' transcription and has an effect on spore formation in Bacillus subtilis. We investigated the role of SpoIIID in mother cell lysis in Bacillus thuringiensis. A ß-galactosidase assay based on the promoter fusions with lacZ indicated that the sigK gene was positively regulated by SpoIIID and σK negatively regulated the expression of sigE. The spoIIID mutant strain exhibited no mother cell lysis in Schaeffer's sporulation medium (SSM) but did in ½ Luria-Bertani (LB) medium. cwlC is an essential hydrolase gene for mother cell lysis. Moreover, the expression of a PcwlC-lacZ fusion in spoIIID mutant was proved to be higher in ½ LB medium than in SSM. HD (ΔspoIIID)(ΔcwlC) mutant was obtained by knocking out the cwlC gene in HD(ΔspoIIID) and displayed no mother cell lysis in both SSM and ½ LB mediums. The deletion of spoIIID decreased the crystal protein production in HD73. The expression of Porf1cry8E and P5014 promoter fusions with lacZ gene in the acrystalliferous HD-(ΔspoIIID) mutant showed similar activity to that in the acrystalliferous HD73- strain before T7 and slightly higher than that in the acrystalliferous HD73- after T7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Cry1Ac production in HD-(ΔspoIIID) directed by the Porf1cry8E and P5014 promoters was at a similar level as that in HD73 wild strain. Altogether, these results suggested that the spoIIID mutant with Porf1cry8E or P5014 promoters could be an alternative delivery system for cry gene expression with no mature spore formation and medium-dependent mother cell lysis.


Assuntos
Bacillus thuringiensis/genética , Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/metabolismo , Bacteriólise , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Deleção de Genes , Fatores de Transcrição/deficiência , Fatores de Transcrição/metabolismo , Bacillus thuringiensis/crescimento & desenvolvimento , Endotoxinas/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas Hemolisinas/metabolismo , Esporos Bacterianos/crescimento & desenvolvimento
8.
Nat Commun ; 10(1): 1407, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926776

RESUMO

RAD51 assembly on single-stranded (ss)DNAs is a crucial step in the homology-dependent repair of DNA damage for genomic stability. The formation of the RAD51 filament is promoted by various RAD51-interacting proteins including RAD51 paralogues. However, the mechanisms underlying the differential control of RAD51-filament dynamics by these factors remain largely unknown. Here, we report a role for the human RAD51 paralogue, SWSAP1, as a novel regulator of RAD51 assembly. Swsap1-deficient cells show defects in DNA damage-induced RAD51 assembly during both mitosis and meiosis. Defective RAD51 assembly in SWSAP1-depleted cells is suppressed by the depletion of FIGNL1, which binds to RAD51 as well as SWSAP1. Purified FIGNL1 promotes the dissociation of RAD51 from ssDNAs. The dismantling activity of FIGNL1 does not require its ATPase but depends on RAD51-binding. Purified SWSAP1 inhibits the RAD51-dismantling activity of FIGNL1. Taken together, our data suggest that SWSAP1 protects RAD51 filaments by antagonizing the anti-recombinase, FIGNL1.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Rad51 Recombinase/metabolismo , Recombinases Rec A/fisiologia , Homologia de Sequência de Aminoácidos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromossomos Humanos/metabolismo , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Células Germinativas/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose , Modelos Biológicos , Ligação Proteica , Recombinases Rec A/genética
9.
Nature ; 567(7749): 530-534, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30814732

RESUMO

T cells expressing chimeric antigen receptors (CAR T cells) targeting human CD19 (hCD19) have shown clinical efficacy against B cell malignancies1,2. CAR T cells have been less effective against solid tumours3-5, in part because they enter a hyporesponsive ('exhausted' or 'dysfunctional') state6-9 triggered by chronic antigen stimulation and characterized by upregulation of inhibitory receptors and loss of effector function. To investigate the function of CAR T cells in solid tumours, we transferred hCD19-reactive CAR T cells into hCD19+ tumour-bearing mice. CD8+CAR+ tumour-infiltrating lymphocytes and CD8+ endogenous tumour-infiltrating lymphocytes expressing the inhibitory receptors PD-1 and TIM3 exhibited similar profiles of gene expression and chromatin accessibility, associated with secondary activation of nuclear receptor transcription factors NR4A1 (also known as NUR77), NR4A2 (NURR1) and NR4A3 (NOR1) by the initiating transcription factor NFAT (nuclear factor of activated T cells)10-12. CD8+ T cells from humans with cancer or chronic viral infections13-15 expressed high levels of NR4A transcription factors and displayed enrichment of NR4A-binding motifs in accessible chromatin regions. CAR T cells lacking all three NR4A transcription factors (Nr4a triple knockout) promoted tumour regression and prolonged the survival of tumour-bearing mice. Nr4a triple knockout CAR tumour-infiltrating lymphocytes displayed phenotypes and gene expression profiles characteristic of CD8+ effector T cells, and chromatin regions uniquely accessible in Nr4a triple knockout CAR tumour-infiltrating lymphocytes compared to wild type were enriched for binding motifs for NF-κB and AP-1, transcription factors involved in activation of T cells. We identify NR4A transcription factors as having an important role in the cell-intrinsic program of T cell hyporesponsiveness and point to NR4A inhibition as a promising strategy for cancer immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias/genética , Neoplasias/imunologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Fatores de Transcrição/metabolismo , Transferência Adotiva , Animais , Antígenos CD19/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Linhagem Celular Tumoral , Cromatina/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Linfócitos do Interstício Tumoral/patologia , Masculino , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Neoplasias/patologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/deficiência , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Receptores de Esteroides/deficiência , Receptores de Esteroides/metabolismo , Receptores dos Hormônios Tireóideos/deficiência , Receptores dos Hormônios Tireóideos/metabolismo , Taxa de Sobrevida , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/deficiência
10.
Pediatr Dermatol ; 36(2): 258-259, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30746751

RESUMO

Neutrophilic panniculitis (NP) with myelodysplasia has been described in adults but not in children. We report a case of NP associated with myelodysplasia in a child with MYSM1 deficiency, a newly described syndrome with primary immunodeficiency (PI), bone marrow failure, and developmental aberrations.


Assuntos
Proteínas de Ligação a DNA/deficiência , Síndromes de Imunodeficiência/diagnóstico , Paniculite/diagnóstico , Fatores de Transcrição/deficiência , Antialérgicos/uso terapêutico , Cetirizina/uso terapêutico , Pré-Escolar , Proteínas de Ligação a DNA/genética , Fármacos Dermatológicos/administração & dosagem , Feminino , Humanos , Síndromes de Imunodeficiência/genética , Furoato de Mometasona/administração & dosagem , Mutação , Paniculite/tratamento farmacológico , Paniculite/genética , Pele/patologia , Fatores de Transcrição/genética
11.
DNA Cell Biol ; 38(4): 314-321, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30762444

RESUMO

The shortage of human hepatocytes continues to be a significant limitation for the widespread application of hepatocyte transplantation and bioartificial liver (BAL) support therapy. Recombinant activation gene 2 (Rag2) and fumarylacetoacetate hydrolase (Fah)-deficient mice could be highly repopulated with human hepatocytes. However, Fah/Rag2-deficient mice can only produce up to 1 × 108 human hepatocytes per mouse. We hypothesized that 2-10 × 1010 human hepatocytes can be produced per Fah/Rag2-deficient pig, which is an adequate supply for hepatocyte transplantation and BAL therapy. In a novel approach, we used stably transfected Cas9 cells and single-guide RNA adenoviruses containing fluorescent reporters to enrich porcine cells with Fah/Rag2 dual gene mutations. This resulted in the construction of Fah/Rag2 double knockout porcine iliac artery endothelial cells, which were subsequently used for generating Fah/Rag2-deficient pigs.


Assuntos
Adenoviridae/genética , Sistemas CRISPR-Cas/genética , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes/métodos , Hidrolases/deficiência , Hidrolases/genética , Animais , Sequência de Bases , Linhagem Celular , Mutação , Suínos , Fatores de Tempo
12.
Gynecol Oncol ; 153(2): 416-424, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30797591

RESUMO

PURPOSE: PARP inhibitor maintenance therapy in platinum sensitive sporadic ovarian cancers improves progression free survival. However, biomarker for synthetic lethality in platinum sensitive sporadic disease is yet to be defined. ERCC1-XPF heterodimer is a key player in nucleotide excision repair (NER) involved in the repair of platinum induced DNA damage. In the current study, we tested whether ERCC1-XPF deficiency would predict synthetic lethality to the PARP inhibitor Olaparib and platinum sensitivity in ovarian cancers. METHODS: ERCC1, XPF and PARP1 protein expression was evaluated in tumors from a cohort of 331 patients treated at Nottingham University Hospitals and correlated to clinicopathological features and survival. Pre-clinically, ERCC1 and XPF was depleted in A2780 (platinum sensitive) and A2780cis (platinum resistant) ovarian cancer cell lines and tested for platinum sensitivity as well as for Olaparib induced synthetic lethality. RESULTS: Low ERCC1 was significantly associated with improved progression free survival (PFS) in patients with ovarian cancers in univariate (p = 0.001) and multivariate (p = 0.002) analysis. In addition, low ERCC1/low XPF (p = 0.003) or low ERCC1/low PARP1 (p = 0.0001) tumors was also linked to better PFS compared to high ERCC1/high XPF or high ERCC1/high PARP1 tumors. Pre-clinically, ERCC1 or XPF depletion not only increased platinum sensitivity but also increased toxicity to Olaparib therapy. Increased sensitivity was associated with DNA double strand breaks (DSBs) accumulation, cell cycle arrest and increased apoptosis. CONCLUSION: The data provide evidence that low ERCC1 is not only a predictor of platinum sensitivity but is also a promising biomarker for Olaparib induced synthetic lethality in ovarian cancers.


Assuntos
Carcinoma Epitelial do Ovário/tratamento farmacológico , Carcinoma Epitelial do Ovário/metabolismo , Proteínas de Ligação a DNA/deficiência , Endonucleases/deficiência , Compostos Organoplatínicos/farmacologia , Ftalazinas/farmacologia , Piperazinas/farmacologia , Carcinoma Epitelial do Ovário/genética , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Endonucleases/biossíntese , Endonucleases/genética , Feminino , Humanos , Imuno-Histoquímica , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/biossíntese , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/genética , Análise Serial de Tecidos , Transfecção
15.
DNA Repair (Amst) ; 74: 70-79, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30606609

RESUMO

DSBs are harmful lesions produced through endogenous metabolism or by exogenous agents such as ionizing radiation, that can trigger genomic rearrangements. We have recently shown that exposure to 2 Gy of X-rays has opposite effects on the induction of Shh-dependent MB in NHEJ- and HR-deficient Ptch1+/- mice. In the current study we provide a comprehensive link on the role of HR/NHEJ at low doses (0.042 and 0.25 Gy) from the early molecular changes through DNA damage processing, up to the late consequences of their inactivation on tumorigenesis. Our data indicate a prominent role for HR in genome stability, by preventing spontaneous and radiation-induced oncogenic damage in neural precursors of the cerebellum, the cell of origin of MB. Instead, loss of DNA-PKcs function increased DSBs and apoptosis in neural precursors of the developing cerebellum, leading to killing of tumor initiating cells, and suppression of MB tumorigenesis in DNA-PKcs-/-/Ptch1+/- mice. Pathway analysis demonstrates that DNA-PKcs genetic inactivation confers a remarkable radiation hypersensitivity, as even extremely low radiation doses may deregulate many DDR genes, also triggering p53 pathway activation and cell cycle arrest. Finally, by showing that DNA-PKcs inhibition by NU7441 radiosensitizes human MB cells, our in vitro findings suggest the inclusion of MB in the list of tumors beneficiating from the combination of radiotherapy and DNA-PKcs targeting, holding promise for clinical translation.


Assuntos
Neoplasias Cerebelares/genética , Reparo do DNA/efeitos da radiação , Meduloblastoma/genética , Neoplasias Induzidas por Radiação/genética , Receptor Patched-1/deficiência , Receptor Patched-1/metabolismo , Animais , Carcinogênese/genética , Carcinogênese/efeitos da radiação , Linhagem Celular Tumoral , Neoplasias Cerebelares/metabolismo , Neoplasias Cerebelares/patologia , Neoplasias Cerebelares/terapia , Dano ao DNA , Reparo do DNA por Junção de Extremidades/efeitos da radiação , DNA Helicases/genética , Proteína Quinase Ativada por DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Relação Dose-Resposta à Radiação , Recombinação Homóloga/efeitos da radiação , Humanos , Meduloblastoma/metabolismo , Meduloblastoma/patologia , Meduloblastoma/terapia , Camundongos , Terapia de Alvo Molecular , Mutação , Neoplasias Induzidas por Radiação/metabolismo , Neoplasias Induzidas por Radiação/patologia , Neoplasias Induzidas por Radiação/terapia , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Risco , Raios X/efeitos adversos
16.
Curr Microbiol ; 76(3): 320-328, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30684026

RESUMO

Pseudomonas aeruginosa, which is a clinically important representative of Pseudomonas spp., has been recognized as causative agent of severe nosocomial infections worldwide. An increase in antibiotic resistance of P. aeruginosa clinical strains could be attributed to their capacity to acquire resistance through mobile genetic elements such as mobile integrons that are present in one-half of multidrug-resistant P. aeruginosa strains. Mobile class 1 integrons are recognized as genetic elements involved in the rapid dissemination of multiple genes encoding for antibiotic resistance. The LexA protein is a major repressor of integrase transcription, but differences in transcription regulation among bacterial species have also been noted. In this study, the promoter activity of class 1 integron integrase gene (intI1) and its variant lacking the LexA binding site in Pseudomonas putida WCS358 wild type, ΔrpoS and ΔpsrA was analysed. The results show that the activity of the intI1 gene promoter decreased in the rpoS and psrA mutants in the stationary phase of growth compared to the wild type, which indicates the role of RpoS and PsrA proteins in the positive regulation of integrase transcription. Additionally, it was determined that the activity of the lexA gene promoter decreased in ΔrpoS and ΔpsrA, and thus, we propose that PsrA indirectly regulates the intI1 gene promoter activity through regulation of lexA gene expression in co-operation with some additional regulators. In this study, intI1 gene expression was shown to be controlled by two major stress response (SOS and RpoS) regulons, which indicates that integrase has evolved to use both systems to sense the cell status.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Integrases/genética , Pseudomonas/fisiologia , Serina Endopeptidases/genética , Fatores de Transcrição/metabolismo , Sítios de Ligação , Fenômenos Fisiológicos Celulares , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Modelos Genéticos , Regiões Promotoras Genéticas , Pseudomonas/genética , Pseudomonas/crescimento & desenvolvimento , Regulon , Deleção de Sequência , Serina Endopeptidases/metabolismo , Fator sigma/deficiência , Fator sigma/genética , Fator sigma/metabolismo , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética
17.
J Immunol ; 202(5): 1573-1581, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30665938

RESUMO

Antibody diversity is initiated by activation-induced deaminase (AID), which deaminates cytosine to uracil in DNA. Uracils in the Ig gene loci can be recognized by uracil DNA glycosylase (UNG) or mutS homologs 2 and 6 (MSH2-MSH6) proteins, and then processed into DNA breaks. Breaks in switch regions of the H chain locus cause isotype switching and have been extensively characterized as staggered and blunt double-strand breaks. However, breaks in V regions that arise during somatic hypermutation are poorly understood. In this study, we characterize AID-dependent break formation in JH introns from mouse germinal center B cells. We used a ligation-mediated PCR assay to detect single-strand breaks and double-strand breaks that were either staggered or blunt. In contrast to switch regions, V regions contained predominantly single-strand breaks, which peaked 10 d after immunization. We then examined the pathways used to generate these breaks in UNG- and MSH6-deficient mice. Surprisingly, both DNA repair pathways contributed substantially to break formation, and in the absence of both UNG and MSH6, the frequency of breaks was severely reduced. When the breaks were sequenced and mapped, they were widely distributed over a 1000-bp intron region downstream of JH3 and JH4 exons and were unexpectedly located at all 4 nt. These data suggest that during DNA repair, nicks are generated at distal sites from the original deaminated cytosine, and these repair intermediates could generate both faithful and mutagenic repair. During mutagenesis, single-strand breaks would allow entry for low-fidelity DNA polymerases to generate somatic hypermutation.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , Região Variável de Imunoglobulina/genética , Uracila-DNA Glicosidase/genética , Animais , Quebras de DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/imunologia , Região Variável de Imunoglobulina/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Uracila-DNA Glicosidase/deficiência , Uracila-DNA Glicosidase/imunologia
18.
Clin Cancer Res ; 25(8): 2523-2536, 2019 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-30538112

RESUMO

PURPOSE: ERCC1/XPF is a DNA endonuclease with variable expression in primary tumor specimens, and has been investigated as a predictive biomarker for efficacy of platinum-based chemotherapy. The failure of clinical trials utilizing ERCC1 expression to predict response to platinum-based chemotherapy suggests additional mechanisms underlying the basic biology of ERCC1 in the response to interstrand crosslinks (ICLs) remain unknown. We aimed to characterize a panel of ERCC1 knockout (Δ) cell lines, where we identified a synthetic viable phenotype in response to ICLs with ERCC1 deficiency. EXPERIMENTAL DESIGN: We utilized the CRISPR-Cas9 system to create a panel of ERCC1Δ lung cancer cell lines which we characterized. RESULTS: We observe that loss of ERCC1 hypersensitizes cells to cisplatin when wild-type (WT) p53 is retained, whereas there is only modest sensitivity in cell lines that are p53mutant/null. In addition, when p53 is disrupted by CRISPR-Cas9 (p53*) in ERCC1Δ/p53WT cells, there is reduced apoptosis and increased viability after platinum treatment. These results were recapitulated in 2 patient data sets utilizing p53 mutation analysis and ERCC1 expression to assess overall survival. We also show that kinetics of ICL-repair (ICL-R) differ between ERCC1Δ/p53WT and ERCC1Δ/p53* cells. Finally, we provide evidence that cisplatin tolerance in the context of ERCC1 deficiency relies on DNA-PKcs and BRCA1 function. CONCLUSIONS: Our findings implicate p53 as a potential confounding variable in clinical assessments of ERCC1 as a platinum biomarker via promoting an environment in which error-prone mechanisms of ICL-R may be able to partially compensate for loss of ERCC1.See related commentary by Friboulet et al., p. 2369.


Assuntos
Proteínas de Ligação a DNA/deficiência , Neoplasias Pulmonares , Cisplatino , Reparo do DNA , Endonucleases/deficiência , Humanos
19.
DNA Repair (Amst) ; 73: 164-169, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30579708

RESUMO

Non-homologous end joining (NHEJ) is a DNA repair pathway that senses, processes and ligates DNA double-strand breaks (DSBs) throughout the cell cycle. During NHEJ, core Ku70 and Ku80 subunits bind DSBs as a heterodimer and promote further recruitment of accessory factors (e.g., PAXX, Mri, DNA-PKcs, Artemis) and downstream core subunits XRCC4 and DNA ligase 4 (Lig4). Inactivation of Ku70 or Ku80 genes in mice results in immunodeficiency and high levels of genomic instability; deletion of individual Dna-pkcs, Xlf, Paxx or Mri genes results in viable mice with no or modest DNA repair defects. However, combined inactivation of either Xlf and Dna-pkcs, or Xlf and Paxx, or Xlf and Mri, leads to synthetic lethality in mice, which correlates with increased levels of apoptosis in the central nervous system. Here, we demonstrated that inactivation of pro-apoptotic factor Trp53 rescues embryonic lethality of Xlf-/-Paxx-/- and Xlf-/-Dna-pkcs-/- double knockout mice. Moreover, combined inactivation of Paxx and Dna-pkcs results in live-born fertile Paxx-/-Dna-pkcs-/- mice indistinguishable from Dna-pkcs-/- knockout controls.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/genética , Inativação Gênica , Mutações Sintéticas Letais , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Animais , Linhagem Celular , Enzimas Reparadoras do DNA/genética , Proteína Quinase Ativada por DNA/deficiência , Proteína Quinase Ativada por DNA/genética , Proteínas de Ligação a DNA/deficiência , Técnicas de Inativação de Genes , Humanos , Camundongos , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética
20.
Cancer Lett ; 444: 136-146, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30579971

RESUMO

Xeroderma pigmentosum group C (XPC) is a well-known DNA damage recognition protein. Defects in XPC lead to carcinogenesis and progression of many human cancers. In the current study, we defined a novel, important role of XPC in preventing centrosome amplification during cisplatin-mediated DNA damage response. From experiments with human bladder cancer tissue, urothelial tissue from Xpc knockout mice and XPC-silenced cell lines, we found that attenuated XPC expression was associated with increased centrosome amplification in human bladder cancer. A significant increase in centrosome amplification was observed in XPC-silenced cells upon cisplatin treatment. XPC deficiency leads to reduced BRCA1 expression via upregulating its transcriptional repressor, Pit-1. The BRCA1 downregulation results in more DNA double strand breaks accumulation and persistent activation of the ATM-Chk1/Chk2 signaling, resulting in a prolonged G2/M arrest during which centrosome can over-duplicate and lead to centrosome amplification. XPC complementation in silenced cells could reduce Pit-1 expression, increase BRCA1 expression and recover the status of centrosome amplification. Our study reveals a new function for XPC in preventing chromosomal instability, providing new information on cancer chemotherapy and potential clinical significance for cancer management.


Assuntos
Proteína BRCA1/antagonistas & inibidores , Centrossomo , Cisplatino/farmacologia , Dano ao DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/fisiologia , Neoplasias da Bexiga Urinária/genética , Animais , Antineoplásicos/farmacologia , Apoptose , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proliferação de Células , Reparo do DNA , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Knockout , Células Tumorais Cultivadas , Neoplasias da Bexiga Urinária/metabolismo , Neoplasias da Bexiga Urinária/patologia
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